干扰Smad3促进山羊脂肪细胞分化

doi:10.11843/j.issn.0366-6964.2020.03.008

摘要/Abstract

摘要： 旨在克隆山羊Smad3的基础上，明确其组织和细胞表达谱，最终阐明干扰Smad3基因对山羊肌内和皮下脂肪细胞分化的影响。本研究选用5只体况良好的1周龄简州大耳羊，空腹24 h后屠宰并采集相应组织和细胞进行试验。利用RT-PCR技术克隆山羊Smad3基因cDNA区序列并进行生物信息学分析，利用实时荧光定量PCR（real-time quantitative PCR，qPCR）技术检测Smad3基因的组织和细胞时序表达水平；并且合成靶向Smad3的siRNA，采用油红O染色从形态学上明确干扰Smad3对山羊前体脂肪细胞成脂分化的影响，利用qPCR检测干扰Smad3对脂肪细胞分化标志基因C/EBPα、C/EBPβ、LPL、SREBP1、AP2、PPARγ、Pref-1、KLF3、KLF4、KLF6、KLF7、KLF8、KLF9、KLF10和KLF15以及Smads相关基因Smad2、Smad4、Smad7和TGF-β1基因mRNA表达水平的影响，探讨可能的作用机制。结果，获得山羊Smad3基因1 449 bp，其中CDS区序列为1 278 bp，编码425个氨基酸；Smad3在山羊各组织中具有广泛表达特性，且在肾脏中的表达水平最高（P<0.01）；Smad3均在山羊肌内和皮下两种脂肪细胞诱导分化的36 h表达量最低，极显著低于在未分化前体脂肪细胞中的表达丰度（P<0.01）；干扰Smad3后发现显著促进了山羊肌内和皮下脂肪细胞中脂滴的聚集，且脂肪细胞分化标志基因、KLF3、KLF4、KLF8、KLF9、KLF10和KLF15的表达水平显著上升（P<0.05），Pref-1的相对表达水平极显著下降（P<0.01），同时干扰Smad3基因下调了Smad2、Smad4和Smad7基因的相对表达水平（P<0.05）。研究结果指出，干扰Smad3促进山羊脂肪细胞分化，且可能通过调控脂肪细胞分化标志基因C/EBPα、C/EBPβ、LPL、SREBP1、AP2、Pref-1、KLF3、KLF4、KLF8、KLF9、KLF10和KLF15等及协同Smad2、Smad4和Smad7的表达来实现的。

关键词: 山羊, Smad3, 基因克隆, RNA干扰, 肌内脂肪细胞, 皮下脂肪细胞

Abstract: The aim of this study was to clone goat Smad3, to clarify its tissue and cell expression profiles, and finally to elucidate the effect of interfering Smad3 gene on the differentiation of goat intramuscular and subcutaneous adipocytes. In this study, 5 one-week-old Jianzhou Big-ear goats in good physical condition were selected, slaughtered after fasted for 24 h, and the corresponding tissues and cells were sampled for testing. The cDNA sequence of goat Smad3 gene was cloned by RT-PCR and analyzed by bioinformatics. Real-time quantitative PCR (qPCR) was used to detect the tissue and cell temporal expression of Smad3 gene. siRNA targeting to Smad3 was designed and synthesized. Oil red O staining was used to detect the effect of interfering Smad3 on adipogenic differentiation of goat preadipocytes, and qPCR was used to detect the effect of interfering Smad3 on the expression levels of adipocyte differentiation marker genes (C/EBPα, C/EBPβ, LPL, SREBP1, AP2, PPARγ, Pref-1, KLF3, KLF4, KLF6, KLF7, KLF8, KLF9, KLF10 and KLF15), Smads-related genes (Smad2, Smad4, Smad7) and TGF-β1 mRNA, with exploring the possible mechanism. The results showed that the goat Smad3 gene was 1 449 bp in length, the CDS region sequence was 1 278 bp, encoding 425 amino acids. Smad3 was widely expressed in goat tissues with the highest expression level in the kidney (P<0.01); Smad3 expression was lowest in goat intramuscular and subcutaneous adipocytes at 36 h of induced differentiation, which was significantly lower than that in undifferentiated precursor adipocytes (P<0.01). The interference of Smad3 significantly promoted the accumulation of lipid droplets in intramuscular and subcutaneous adipocytes of goats, the expression level of adipocyte differentiation marker genes, KLF3, KLF4, KLF8, KLF9, KLF10 and KLF15 were significantly increased (P<0.05), and the relative expression level of Pref-1 was extremely significantly decreased (P<0.01). At the same time, the interference of Smad3 gene down-regulated the relative expression levels of Smad2, Smad4 and Smad7 genes (P<0.05). The results indicate that interference of Smad3 promotes the differentiation of goat adipocytes by regulating the expression of adipocyte differentiation marker genes LPL, SREBP1, AP2, C/EBPα, C/EBPβ, Pref-1, KLF3, KLF4, KLF8, KLF9, KLF10 and KLF15 cooperating with Smad2, Smad4 and Smad7.

Key words: goat, Smad3, gene cloning, RNA interference, intramuscular adipocytes, subcutaneous adipocytes

中图分类号:

崔胜, 林亚秋, 许晴, 朱江江, 王永. 干扰Smad3促进山羊脂肪细胞分化[J]. 畜牧兽医学报, 2020, 51(3): 475-489.

CUI Sheng, LIN Yaqiu, XU Qing, ZHU Jiangjiang, WANG Yong. Interfering Smad3 Promotes Goat Adipocyte Differentiation[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(3): 475-489.

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